- Email: [email protected]
A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease
Protein Expression and Purification 128 (2016) 86e92
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
A simple method for recombinant protein purification using selfassembling peptide-tagged tobacco etch virus protease Guang-Ya Li, Zhen-Zhen Xiao, Hui-Peng Lu, Yang-Yang Li, Xiao-Hui Zhou, Xiao Tan, Xin-Yu Zhang, Xiao-Li Xia, Huai-Chang Sun* College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2016 Received in revised form 15 August 2016 Accepted 17 August 2016 Available online 19 August 2016
Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptidetagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-g (His-BoIFN-g) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 104 units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-g (rBoIFNg) was purified to 98.3% purity with 63% recovery. The rBoIFN-g had an antiviral activity of 1.6 103 units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification. © 2016 Elsevier Inc. All rights reserved.
Keywords: Tobacco etch virus protease Self-assembling peptide Active protein aggregate Protein purification
1. Introduction Recombinant proteins are often fused to various tags to facilitate their detection and purification, increase yields and/or enhance solubility. However, it is almost always desirable to obtain the native protein free of fusion partner since most fusion tags are expected to interfere with the structural studies and/or the biological activity of the target protein [1,2]. This is achieved commonly by introducing a protease recognition sequence and then cleaving at the junction between the fusion tag and the target protein. The commonly used proteases for this purpose include thrombin, factor Xa, enterokinae and tobacco etch virus protease (TEVp). Among them, TEVp is a popular protease targeting the recognition sequence ENLYFQG/S with several advantages, including stringent sequence specificity, overproduction in E. coli and adaptability to different buffer conditions [3e5]. As the tool for fusion tag removal, wild type TEVp suffers from a
* Corresponding author. E-mail addresses: [email protected], [email protected] (H.-C. Sun). http://dx.doi.org/10.1016/j.pep.2016.08.013 1046-5928/© 2016 Elsevier Inc. All rights reserved.
few intrinsic defects, including poor expression, low solubility in E. coli and self-activation to generate a truncated enzyme with diminished activity [6]. Therefore, various approaches have been investigated to overcome these limitations and several TEVp mutants have been generated, including S219V or S219N mutant with significantly reduced auto-proteolysis, L56V/S135G double mutant, T17S/N68D/I77V triple mutant and T17S/L56V/N68D/I77V/S135G quintuple mutant with improved solubility and thermal stability [6e9]. Although these TEVp mutants have been expressed in E. coli as 6 His, glutathione S-transferase (GST) or lectin fusion protein with high enzyme activity, their purification from bacterial extracts and removal from the cleavage reactions require expensive affinity chromatographic columns [10e12]. Bacterially expressed inclusion bodies (IBs) are generally considered to be mis-folded protein aggregates without biological activities. However, recent studies have revealed that some protein aggregates, known as non-classical or active IBs, contain correctly folded and functionally active proteins [13e15]. More recently, several self-assembling peptides have been used as the efficient inducer of active protein aggregates [16e18]. Compared with other aggregating fusion partners, self-assembling peptides are much
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
87
smaller in size and structurally simple, and have higher “pulldown” efficiencies. For example, self-assembling peptide ELK16 (LELELKLK)2 can spontaneously form b-sheet structure in aqueous solution, and function as an inducer of active protein aggregates when terminally attached to model proteins, including lipase A, amadoriase II, b-xylosidase and green fluorescent protein [17]. Therefore, self-assembling peptide-induced active aggregation is expected to have attractive application potentials in biotechnology, including in-situ immobilization of carrier-free enzymes, production of toxic proteins, and protein expression and purification [18]. In this study, we developed a simple method for recombinant protein purification by using self-assembling peptide ELK16-tagged TEVp (ELK16-TEVp). The proof of concept was provided by purifying recombinant bovine interferon-g (rBoIFN-g), which has been used to control many infectious cattle diseases [19e21]. We demonstrated that the ELK16-TEVp could be expressed as active protein aggregates and purified to a high purity with high protease activity.
ELK16-TEVp was purified using the method for active protein aggregate purification [17] with slight modification. For the pilot experiment, the expression of ELK16-TEVp was induced with 0.2 mM IPTG for 6 h at 30 C. Cells were harvested from 50 mL culture, and lyzed in 5 mL lysis buffer by sonication treatment. ELK16-TEVp was precipitated by centrifugation for 10 min at different centrifugation forces at 4 C. For the larger scale purification, the expression of ELK16-TEVp was induced for 16 h at optimized conditions. A total of 4 g wet weight cells was harvested from 500 mL culture and lyzed in 50 mL lysis buffer by sonication treatment. ELK16-TEVp was precipitated by centrifugation for 10 min at optimized centrifugation force at 4 C. After washing two times in 50 mL lysis buffer containing 0.5% Triton X-100, the purified ELK16-TEVp was washed one time with 50 mL TEVp cleavage buffer (50 mM Tris-HCl, pH8.0, 0.5 mM EDTA, 1 mM DTT), and dissolved in 5 mL TEVp cleavage buffer.
2. Materials and methods
2.5. Purification of His-BoIFN-g
2.1. Vector construction
His-BoIFN-g was purified using Ni-NTA Agarose Column (Qiagen, USA) under native conditions by following the product instruction. One milliliter of Ni-NTA slurry (50%) was loaded into a column (1 mL), and equilibrated with 8 mL lysis buffer. The column was loaded with 5 mL cleared bacterial lysate from 50 mL culture (0.95 g wet weight), and washed two times with 8 mL washing buffer containing 40 mM imidazole. His-BoIFN-g was eluted with 5 mL elution buffer containing 250 mM imidazole. The purified protein was dialyzed in 25 mL TEVp cleavage buffer for 9 h at 4 C with 3 buffer changes. After centrifugation for 10 min at 16,000g at 4 C, His-BoIFN-g was diluted to 500 mg/mL in TEVp cleavage buffer.
To construct an N-terminal self-assembling peptide fusion expression vector, the coding sequence for ELK16 peptide flanked by 17 proline (P) or threonine (T) rigid linkers [17] was adapted to E. coli codon usage using JAVA Codon Adaption Tool [22]. The synthetic sequence was cloned into pET-30a vector (Novagen, WI, USA) as a BglII/KpnI segment, and the resultant vector was called pET-P16P. To construct an ELK16-TEVp expression vector, the coding sequence for T17S/L56V/N68D/I77V/S135G quintuple mutant of TEVp [9] was adapted to E. coli codon usage, and the synthetic sequence was cloned into pET-P16P vector as an EcoRI/XhoI segment. The resultant vector was called pP16P-TEVp (Fig. 1a). To construct a His-BoIFN-g expression vector, the coding sequence for the mature peptide of BoIFN-g with a TEVp recognition sequence at the 50 end was amplified from pGEX-BoIFN-g vector [23] by PCR using rTaq DNA polymerase (TaKaRa, Dalian, China), and the forward primer FP (50 -GAGGATCCGAAAACCTGTACTTCCAGGGTCAGGGCCAATTTTTTAGAGAA-30 ) and reverse primer RP (50 -TACTCGAGTTACGTTGATGCTCTCCGGCC-30 ). The PCR product was cloned into pET-30a vector as a BamHI/XhoI segment, and the resultant vector was called pET-BoIFN-g (Fig. 2a). 2.2. Protein expression pP16P-TEVp or pET-BoIFN-g vector was transformed into E. coli strain BL21 (DE3), and the Luria broth culture supplemented with 50 mg/mL kanamycin was grown overnight at 37 C in an orbital shaker. The cell culture was diluted (1:100) in 2 YT medium (10 g yeast extract, 16 g tryptone, 5 g NaCl/L) supplemented with the same antibiotic, and grown to OD600 0.6 at 37 C. The expression of fusion protein was induced with different concentrations of IPTG for different times at different temperatures.
2.4. Purification of ELK16-TEVp
2.6. Protease cleavage and target protein recovery The protease cleavage reaction (100 mL) consisted of different amounts of ELK16-TEVp or GST-TEVp (BBI Life Sciences, USA) and His-BoIFN-g in the presence or absence of different additives. The cleavage reaction was carried out for 1 or 2 h at different pH at different temperatures. After cleavage, ELK16-TEVp was removed by centrifugation for 10 min at 16,000g at 4 C. Following the absorption with an equal volume of 50% Ni-NTA agarose slurry (Qiagen, USA) for 30 min at 4 C, His-tag was removed by an additional round of centrifugation. The cleavage reaction was followed by 12% or 15% SDS-PAGE analysis. After Commassie blue staining, the protein bands of interest were scanned using the Image Lab™ Software associated with GelDoc XR System (Bio-Rad, USA). The cleavage efficiency (%) was calculated according to the percent ratio of BoIFN-g/His-BoIFN-g band densities. One unit of ELK16-TEVp was defined as 85% cleavage of 3 mg substrate within 1 h at optimized conditions. The purified BoIFN-g was submitted to 1 cycle of Triton X-114 isothermal extraction to remove residual endotoxin [24]. The purified protein was mixed with 1% Triton X114 (final concentration), incubated for 10 min on ice and then 5 min at 37 C. After centrifugation for 1 min at 12,000g at 30 C, the upper aqueous phase was collected for antiviral assay.
2.3. Cell lysis 2.7. Antiviral assay After IPTG induction, cells were harvested by centrifugation for 10 min at 6,000g at 4 C, washed two times in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH7.2), and disrupted in the lysis buffer by sonication treatment (40 w, 10 s, 20 s intervals, 5 min). After centrifugation for 10 min at 6,000g (ELK16-TEVp) or 12,000g (His-BoIFN-g), the crude cell extract was collected for protein purification.
The antiviral activity of purified rBoIFN-g against vesicular stomatitis virus (VSV) was determined on MDBK cells (ATCC, USA) as described previously [25]. Briefly, the cells were seeded on 96-well plates, grown to 90% confluence and serial dilutions of rBoIFN-g were added in triplicates. After incubation for 24 h at 37 C, an optimal concentration (100 TCID50) of VSV was added. After
88
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
incubation for an additional 24 h, the antiviral activity was calculated according to the highest dilution of rBoIFN-g with 50% cytopathic inhibitory effect. 3. Results 3.1. Vector construction Since all of the reported recombinant TEV proteases are expressed with an N-terminal fusion tag, we constructed an Nterminal ELK16 fusion expression vector by cloning the peptide coding sequence downstream of the S-tag in pET-30a vector. Sequence analysis showed that the coding sequence for the quintuple TEVp mutant had an E. coli codon adaption index (CAI) of only 0.29. Therefore, the protease coding sequence was adapted to E. coli codon usage (CAI 1.0), and cloned in frame with the ELK16 coding sequence in the pET-P16P vector (Fig. 1a). To provide the proof of concept for protein purification using ELK16-TEVp, the coding sequence for BoIFN-g with a TEVp recognition sequence was cloned into pET-30a vector and expressed a His-BoIFN-g fusion protein (Fig. 2a). 3.2. Expression and purification of ELK16-TEVp Previous studies have shown that ELK16 fusion proteins can be expressed an active protein aggregates at 30 C. Therefore, we induced ELK16-TEVp expression first with different concentrations of IPTG at 30 C. SDS-PAGE analysis showed that, compared to the
Fig. 2. Expression and purification of His-BoIFN-g. (a) The schematic structure of HisBoIFN-g expression vector. (b) Induction of His-BoIFN-g expression with different concentrations of IPTG for 6 h at 37 C; (c) Purification of His-BoIFN-g. After induction with 0.1 mM IPTG for 16 h at 20 C, cells were harvested from 250 mL culture, and HisBoIFN-g was purified using Ni-NTA column under native conditions. The protein samples were analyzed on 12% SDS-PAGE. Lane 1: total cell extract; lane 2: insoluble fraction; lane 3: soluble fraction; lane 4: purified His-BoIFN-g.
uninduced cell lysate, an expected 37-kDa extra protein band (5.5kDa PT-ELK16-PT þ 26.3-kDa TEVp þ 5.2-kDa vector sequence) was present in IPTG-induced cell lysate, and 0.2 mM IPTG was sufficient to induce the high level expression (Fig. 1b). About 90% of ELK16TEVp could be precipitated by centrifugation force as low as 1,000g, the amount of which was increased slightly as the increase of centrifugation force (Fig. 1c). Since more than 90% of ELK16-TEVp could be precipitated by lower centrifugation forces, the fusion protein was precipitated at a larger scale (500 mL culture) by centrifugation at 6,000g. After washing two times in 0.5% Triton X100 and one time in TEVp cleavage buffer, quantitative SDS-PAGE analysis showed that ELK16-TEVp was purified to 91% purity with 92% recovery (Fig. 1d). 3.3. Expression and purification of His-BoIFN-g
Fig. 1. Expression and purification of ELK16-TEVp. (a) The schematic structure ELK16TEVp expression vector. (b) Induction of ELK16-TEVp expression at 30 C with different concentrations of IPTG; (c) Precipitation of ELK16-TEVp at different centrifugation forces; (d) Purification of ELK16-TEVp. After induction with 0.2 mM IPTG for 16 h at 20 C, cells were harvested from 500 mL cell culture, and ELK16-TEVp was precipitated by centrifugation at 6,000g, washed two times in 0.5% Triton X-100 and one time in TEVp cleavage buffer. The protein samples were analyzed on 12% SDS-PAGE. Lane 1: total cell extract; lane 2: soluble fraction; lane 3: insoluble fraction; lane 4: protein aggregates washed one time in Triton X-100; lane 5: protein aggregates washed two times in Triton X-100; lane 6: protein aggregates washed one more time in TEVp cleavage buffer.
First, the expression of His-BoIFN-g was induced with different concentrations of IPTG for 6 h at 37 C. SDS-PAGE analysis showed that, compared to the uninduced cell lysate, an expected extra 23kDa protein band (5.4-kDa His- and S-tags þ 16-kDa target protein) was revealed in the IPTG-induced cell lysate, 50% of which was present in the insoluble fraction. The highest level expression could be induced with 0.1 mM IPTG (Fig. 2b). Then, the expression of HisBoIFN-g was induced with 0.1 mM IPTG for 16 h at 20 C, and purified from 250 mL culture using Ni-NTA Agarose column under the native condition. Quantitative SDS-PAGE analysis showed that HisBoIFN-g was purified to 87.5% purity with a recovery of 88.0% (Fig. 2c). 3.4. Optimization of the temperature for active ELK16-TEVp expression Since high temperature is one of the main reasons for classic
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
89
with the highest cleavage rate (93%) at pH8.0 at 37 C (Fig. 4a). In consideration of the potential detrimental effect of higher temperature (37 C) on the protease stability, the following cleavage reaction was performed at 30 C. Quantitative SDS-PAGE analysis showed that the cleavage efficiency of ELK16-TEVp was also pHdependent with the highest cleavage rate (91%) at pH8.0 (Fig. 4b). 3.6. Determination and comparison ELK16-TEVp activity
Fig. 3. Optimization of the temperature for active ELK16-TEVp expression. The expression of ELK16-TEVp was induced with 0.2 mM IPTG for 6 h at different temperatures. The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of purified ELK16-TEVp for 1 h at H8.0 at 30 C. The cleavage reactions were analyzed on 12% SDS-PAGE, and the band densities of BoIFN-g and His-BoIFN-g were scanned for the calculation of cleavage rates after Coomassie blue staining.
inclusion body formation, we optimized the temperature for more active ELK16-TEVp expression. First, the expression of ELK16-TEVp was induced at different temperatures and purified as described. Then, 500 ng purified ELK16-TEVp was used to cleave 30 mg HisBoIFN-g under the standard conditions (pH8.0, 30 C, 1 h). Quantitative SDS-PAGE analysis showed that the cleavage efficiency was decreased as the increase of ELK16-TEVp expression temperature, with the highest cleavage rate (64%) at expression temperature of 20 C (Fig. 3). Therefore, the following experiments were performed using the ELK16-TEVp expressed at 20 C.
First, ELK16-TEVp activity was determined by cleaving a fixed amount (3 mg) of His-BoIFN-g with different amounts of ELK16TEVp under standard conditions. Quantitative SDS-PAGE analysis showed that the cleavage efficiency was ELK16-TEVp dosedependent with a cleavage rate of almost 100% at 150 ng. By using 85% cleavage of 3 mg substrate for the unit (U) definition, ELK16-TEVp activity was calculated to be 1U/75 ng or 1.3 104 U/ mg protein (Fig. 5a). Then, the same amount (4 U) of ELK16- or GSTTEVp was used to cleave 30 mg His-BoIFN-g under standard conditions. Quantitative SDS-PAGE analysis showed that ELK16-TEVp had a cleavage rate of 84% (1 h) or almost 100% (2 h), which was much higher than that (30% or 88%) of GST-TEVp (Fig. 5b). 3.7. Effects of additives on ELK16-TEVp activity It is generally accepted that high concentrations of salts, elutants (e.g., imidazole) and detergents (e.g., urea and SDS) in protein purification buffers can affect enzyme activities, including TEVp. To detect the effects of different additives on ELK16-TEVp activity, 30 mg His-BoIFN-g was cleaved with 500 ng EKL16-TEVp in the presence of different additives. Quantitative SDS-PAGE analysis showed that the cleavage rate was enhanced slightly in the presence of 50 mM NaCl, but inhibited significantly by higher concentration (250 mM) of NaCl (Fig. 6a). Unlike the relatively stable activity in the presence of different concentrations of imidazole (Fig. 6b), ELK16-TEVp activity was inhibited completely by higher concentrations of urea (2 M) or SDS (0.5%) (Fig. 6c and d). 3.8. Purification of rBoIFN-g using ELK16-TEVp
3.5. Optimization of the conditions for ELK16-TEVp cleavage To optimize the conditions for substrate cleavage, 30 mg HisBoIFN-g was cleaved with 500 ng ELK16-TEVp for 1 h at different pH at different temperatures. Quantitative SDS-PAGE analysis showed that the cleavage efficiency was temperature-dependent
To provide the proof of concept for protein purification using ELK16-TEVp, 30 mg His-BoIFN-g was cleaved with 500 ng EKL16TEVp under standard conditions. SDS-PAGE analysis shows that His-BoIFN-g was cleaved efficiently, and ELK16-TEVp was removed completely from the cleavage reaction by centrifugation. After
Fig. 4. Optimization of the conditions for ELK16-TEVp cleavage. (a) The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of ELK16-TEVp for 1 h at pH8.0 at different temperatures; (b) His-BoIFN-g was cleaved with ELK16-TEVp for 1 h at different pH at 30 C. The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
90
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
Fig. 5. Comparison of ELK16-TEVp activity with GST-TEVp. (a) The same amount (3 mg) of His-BoIFN-g was cleaved with different amounts of ELK16-TEVp for 1 h at pH8.0 at 30 C. The dashed lines indicate the amount of ELK16-TEVp required for 85% cleavage of 3 mg His-BoIFN-g; (b) His-BoIFN-g was cleaved with the same amount (4 U) of ELK16- TEVp or GSTTEVp for 1 or 2 h at pH8.0 at 30 C. The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
Fig. 6. Effects of additives on ELK16-TEVp activity. The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of ELK16-TEVp for 1 h at pH 8.0 at 30 C in the presence of different concentrations of NaCl (a), imidazole (b), urea (c) or SDS (d). The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
absorption with Ni-NTA beads and an additional round of centrifugation, rBoIFN-g was purified to 98.3% purity with 63% recovery (Fig. 7). The performance of ELK16-TEVp for rBoIFN-g purification is summarized in Table 1.
3.9. Antiviral activity of rBoIFN-g After isothermal extraction with Triton X-114, cytopathic inhibition assay showed that the purified rBoIFN-g had an antiviral activity of 1.6 103 U/mg protein against VSV without overt cytotoxicity (Table 2).
4. Discussion TEVp is a popular protease for recombinant protein purification due to its high cleavage efficiency and stringent sequence specificity. Although several recombinant TEV proteases have been Table 1 Performance of purification of rBoIFN-g using ELK16-TEVp. Purification step
Fig. 7. Purification of rBoIFN-g using ELK16-TEVp. A total amount (30 mg) of His-BoIFNg was cleaved with 500 ng ELK16-TEVp for 1 h at pH8.0 at 30 C. After cleavage, ELK16TEVp was removed by centrifugation at 16,000g, and the released His-tag was removed by absorption with Ni-NTA and an additional centrifugation. The protein samples were analyzed on 15% SDS-PAGE. Lane 1: His-BoIFN-g before cleavage; lane 2: cleavage product before ELK16-TEVp removal; lane 3: cleavage product after ELK16-TEVp removal; lane 4: purified rBoIFN-g after His-tag removal.
ELK16-TEVp Cell extract Centrifugation (pellet) Triton X-100 wash Cleavage buffer wash His-BoIFN-g Cell extract Affinity purification After tag cleavage Purified BoIFN-g
Purity (%)
Recovery (%)
Yield (mg/L cell culture)a
26.6 72.3 86.6 91.0
100 95 94 92
132 125 124 121
35.1 87.5 57.6 98.3
100 88 79 63
80 70 63 50
a A total of 8.0 or 9.5 g wet weight cells/L culture was used for purification of ELK16-TEVp or His-BoIFN-g.
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
91
Table 2 Antiviral activity of rBoIFN-g purified using ELK16-TEVp. Group
VSV challenge
rBoIFN-g
100 TCID50
Positive controlb Negative controlc
100 TCID50
a b c
Triplication
1 2 3 þ
CPE50 with indicated dilution of rBoIFN-ga 21
22
23
24
25
26
27
28
29
210
þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
The concentration of stock rBoIFN-g solution was 0.25 mg/mL. The positive control represents the cells were infected with VSV in the absence of rBoIFN-g. The negative control represents the cells were treated with rBoIFN-g only.
reported, most of them are expressed as His- or GST-tagged proteins which require affinity columns for purification and/or removing from the cleavage reactions [10e12]. Recent studies have shown that some self-assembling peptides can act as the inducer of active protein aggregates and their fusion proteins can be purified by centrifugation with high “pull-down” efficiencies [16e18]. In this study, we developed a simple method for recombinant protein purification by using ELK16-TEVp and provided the proof of concept by purification of rBoIFN-g. Among the four self-assembling peptides used in protein purification, all of them are used as a C-terminal fusion tag [16e18]. However, all of the reported TEV proteases are expressed with an Nterminal fusion tag [10e12]. In our pilot experiment, we constructed an N-terminal ELK16 fusion expression vector by cloning the peptide coding sequence into pET-30a vector as an NdeI and BglII segment, but failed to express ELK16-TEVp under all conditions tested. Since the peptide coding sequence was only 51-bp away from the ATG codon in pET-30a vector, we speculated that the fusion protein translation may be interrupted by the proximal N-terminal secondary structure formed by ELK16, which is a selfcomplementary b peptide. To confirm this hypothesis, in this study we separated ELK16 coding sequence with two PT linkers, and cloned the coding sequence more distant (204 bp) from the translation start codon in pET30a vector. Our expression experiment showed that ELK16-TEVp was expressed not only efficiently, but formed active protein aggregates as well. Our experimental data suggest the following advantageous use of ELK16-TEVp for recombinant protein purification. First, ELK16TEVp could be expressed efficiently in E. coli, the expression level (132 mg/L culture) of which was higher than that of other fusiontagged TEVp [7,12]. This may be contributed the factor that the ELK16-TEVp was a quintuple mutant which has improved expression level, solubility and/or stability [9,10]. In addition, the higher expression level may be due to the use of E. coli codon-adapted TEVp coding sequence for expression. Unlike the wild type TEVp that can cleave itself to produce truncated enzymes with diminished activities [6], only single ELK16-TEVp protein band was detected before and after purification, confirming the resistance of quintuple TEVp mutant to auto-activation [6,8,9]. Second, ELK16TEVp could be purified to high purity (91%) with high recovery (92%) by centrifugation due to its expression as active protease aggregates. The first indication for this was the sticky nature of the recombinant bacterial extract. The second evidence was that ELK16-TEVp could be precipitated by centrifugation force as low as 1,000g, which was much lower than that (12,000g) for precipitation of classic inclusion bodies. The precipitation of ELK16-TEVp was neither salt- nor temperature-dependent since it could be precipitated at 4 C or room temperature in buffers with or without 50 mM NaCl. The purification process could be accomplished within 1 h without the need of expensive agents and special equipment. Third, ELK16-TEVp was highly active with almost 100%
cleavage efficiency (2 h) which was significantly higher than that (88%) of GST-TEVp. This may be explained by the simple structure and small size of ELK16 peptide [16], which was favorable for TEVp expression and/or folding. More importantly, unlike other fusiontagged TEVp which requires an extra affinity purification step to remove from the cleavage reaction, ELK16-TEVp could be removed from the cleavage reaction by centrifugation without the need of buffer change. The Ni-NTA absorption step used in this study was to remove the His-tag, but not ELK16-TEVp. Further studies are under the way to improve the purification system by expressing the target proteins as ELK16 fusions. If successful, both ELK16-TEVp and cleaved ELK16-tag could be removed from the cleavage reactions by single-step centrifugation. Finally, by using ELK16-TEVp, rBoIFN-g could be purified to high purity (98.3%) with high recovery (63%), which was similar or higher than that of chromatographic purification methods [19e21]. One of several advantages of TEVp is its adaptability to different buffer conditions. In this study, for example, ELK16-TEVp activity remained relatively stable at temperatures ranging from 4 C to 40 C, and at pH ranging from 6.5 to 9.5, which was similar to other TEV proteases. Interestingly, ELK16-TEVp activity was enhanced slightly by a low concentration (50 mM) of NaCl, but inhibited significantly by higher concentrations (100 mM) of NaCl. ELK16TEVp activity was not affected in the presence of different concentrations of imidazole, indicating its needless to remove from the protein elutants for ELK16-TEVp cleavage. Finally, ELK16-TEVp activity was affected significantly by higher concentrations of urea (2 M) or SDS (0.5%), which was different from MBP- or Histagged TEVp [10]. 5. Conclusions In this paper we report a novel method for recombinant protein purification using self-assembling peptide ELK16-TEVp. Since its expression as active protein aggregates, ELK16-TEVp could be purified to a high purity and removed from the cleavage reaction by centrifugation. The proof of concept was provided by purifying rBoIFN-g. Our data suggest ELK16-TEVp may become a universal tool for recombinant protein purification. Acknowledgments This work was supported by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and the fund from Jiangsu Key Laboratory of Zoonosis. References [1] A. Malhotra, Tagging for protein expression, Methods Enzymol. 463 (2009) 239e258. [2] D. Walls, S.T. Loughran, Tagging recombinant proteins to enhance solubility and aid purification, Methods Mol. Biol. 681 (2011) 151e175.
92
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
[3] D.S. Waugh, An overview of enzymatic reagents for the removal of affinity tags, Protein Expr. Purif. 80 (2011) 283e293. [4] J.A. Daros, M.C. Schaad, J.C. Carrington, Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants, J. Virol. 73 (1999) 8732e8740. [5] R.B. Kapust, J. Tozser, T.D. Copeland, D.S. Waugh, The P1' specificity of tobacco etch virus protease, Biochem. Biophys. Res. Commun. 294 (2002) 949e955. [6] R.B. Kapust, J. Tozer, J.D. Fox, D.E. Anderson, S. Cherry, T.D. Copeland, D.S. Waugh, Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency, Protein Eng. 14 (2001) 993e1000. [7] S. van den Berg, P.A. Lofdahl, T. Hard, H. Berglund, Improved solubility of TEV protease by directed evolution, J. Biotechnol. 121 (2006) 291e298. [8] L.D. Cabrita, D. Gilis, A.L. Robertson, Y. Dehouck, M. Rooman, S.P. Bottomley, Enhancing the stability and solubility of TEV protease using in silico design, Protein Sci. 16 (2007) 2360e2367. [9] L. Wei, X. Cai, Z. Qi, L. Rong, B. Cheng, J. Fan, In vivo and in vitro characterization of TEV protease, Protein Expr. Purif. 83 (2012) 157e163. [10] C. Sun, J. Liang, R. Shi, X. Gao, R. Zhang, F. Hong, Q. Yuan, S. Wang, Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives, Protein Expr. Purif. 82 (2012) 226e231. [11] K. Zhu, X. Zhou, Y. Yan, H. Mo, Y. Xie, B. Cheng, J. Fan, Cleavage of fusion proteins on the affinity resins using the TEV protease variant, Protein Expr. Purif. (2016) 30023e30027. S1046-5928. [12] X.J. Li, J.L. Liu, D.S. Gao, W.Y. Wan, X. Yang, Y.T. Li, H.T. Chang, L. Chen, C.Q. Wang, J. Zhao, Single-step affinity and cost-effective purification of recombinant proteins using the sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner, Protein Expr. Purif. 119 (2016) 51e56. [13] E. Garcia-Fruitos, N. Gonzalez-Montalban, M. Morell, A. Vera, R.M. Ferraz, A. Aris, S. Ventura, A. Villaverde, Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins, Microb. Cell Fact. 4 (2005) 27. [14] S. Jevsevar, V. Gaberc-Porekar, I. Fonda, B. Podobnik, J. Grdadolnik, V. Menart,
[15] [16]
[17]
[18] [19] [20]
[21]
[22]
[23] [24]
[25]
Production of nonclassical inclusion bodies from which correctly folded protein can be extracted, Biotechnol. Prog. 21 (2005) 632e639. J.P. Arie, M. Miot, N. Sassoon, J.M. Betton, Formation of active inclusion bodies in the periplasm of Escherichia coli, Mol. Microbiol. 62 (2005) 427e437. X. Wang, B. Zhou, W. Hu, Q. Zhao, Z. Lin, Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8, Microb. Cell Fact. 14 (2015) 88. W. Wu, L. Xing, B. Zhou, Z. Lin, Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli, Microb. Cell Fact. 15 (2011) 9. B. Zhou, L. Xing, W. Wu, X.E. Zhang, Z. Lin, Small surfactant-like peptides can drive soluble proteins into active aggregates, Microb. Cell Fact. 11 (2012) 10. T. Kashima, A. Morishita, H. Iwata, K. Maeda, T. Inoue, Expression of bovine cytokines in Escherichia coli, J. Vet. Med. Sci. 61 (1999) 171e173. K. Murakami, A. Uchiyama, T. Kokuho, Y. Mori, H. Sentsui, T. Yada, M. Tanigawa, A. Kuwano, H. Nagaya, S. Ishiyama, H. Kaki, Y. Yokomizo, S. Inumaru, Production of biologically active recombinant bovine interferongamma by two different baculovirus gene expression systems using insect cells and silkworm larvae, Cytokine 13 (2001) 18e24. D.N. Wedlock, E.E. Doolin, N.A. Parlane, S.J. Lacy-Hulbert, M.W. Woolford, B.M. Buddle, Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils, J. Dairy Res. 67 (2000) 189e197. A. Grote, K. Hiller, M. Scheer, R. Munch, B. Nortemann, D.C. Hempel, D. Jahn, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host, Nucl. Acids Res. 33 (2005) 526e531. J.J. Xu, A.J. Qin, Y.L. Liu, W.J. Jin, R.F. Li, Cloning of cDNA for bovine interferon-g and its expression in E. coli, J. Yangzhou Univ. 24 (2003) 5e10 (In Chinese). R. Ma, J. Zhao, H.C. Du, S. Tian, L.W. Li, Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction, Anal. Biochem. 424 (2012) 124e126. B.L. Osborn, H.S. Olsen, B. Nardelli, J.H. Murray, J.X. Zhou, A. Garcia, G. Moody, L.S. Zaritskaya, C. Sung, Pharmacokinetic and pharmacodynamic studies of a human serum albumin -interferon-alpha fusion protein in cynomolgus monkeys, J. Pharmacol. Exp. Ther. 303 (2002) 540e548.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
A simple method for recombinant protein purification using selfassembling peptide-tagged tobacco etch virus protease Guang-Ya Li, Zhen-Zhen Xiao, Hui-Peng Lu, Yang-Yang Li, Xiao-Hui Zhou, Xiao Tan, Xin-Yu Zhang, Xiao-Li Xia, Huai-Chang Sun* College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2016 Received in revised form 15 August 2016 Accepted 17 August 2016 Available online 19 August 2016
Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptidetagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-g (His-BoIFN-g) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 104 units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-g (rBoIFNg) was purified to 98.3% purity with 63% recovery. The rBoIFN-g had an antiviral activity of 1.6 103 units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification. © 2016 Elsevier Inc. All rights reserved.
Keywords: Tobacco etch virus protease Self-assembling peptide Active protein aggregate Protein purification
1. Introduction Recombinant proteins are often fused to various tags to facilitate their detection and purification, increase yields and/or enhance solubility. However, it is almost always desirable to obtain the native protein free of fusion partner since most fusion tags are expected to interfere with the structural studies and/or the biological activity of the target protein [1,2]. This is achieved commonly by introducing a protease recognition sequence and then cleaving at the junction between the fusion tag and the target protein. The commonly used proteases for this purpose include thrombin, factor Xa, enterokinae and tobacco etch virus protease (TEVp). Among them, TEVp is a popular protease targeting the recognition sequence ENLYFQG/S with several advantages, including stringent sequence specificity, overproduction in E. coli and adaptability to different buffer conditions [3e5]. As the tool for fusion tag removal, wild type TEVp suffers from a
* Corresponding author. E-mail addresses: [email protected], [email protected] (H.-C. Sun). http://dx.doi.org/10.1016/j.pep.2016.08.013 1046-5928/© 2016 Elsevier Inc. All rights reserved.
few intrinsic defects, including poor expression, low solubility in E. coli and self-activation to generate a truncated enzyme with diminished activity [6]. Therefore, various approaches have been investigated to overcome these limitations and several TEVp mutants have been generated, including S219V or S219N mutant with significantly reduced auto-proteolysis, L56V/S135G double mutant, T17S/N68D/I77V triple mutant and T17S/L56V/N68D/I77V/S135G quintuple mutant with improved solubility and thermal stability [6e9]. Although these TEVp mutants have been expressed in E. coli as 6 His, glutathione S-transferase (GST) or lectin fusion protein with high enzyme activity, their purification from bacterial extracts and removal from the cleavage reactions require expensive affinity chromatographic columns [10e12]. Bacterially expressed inclusion bodies (IBs) are generally considered to be mis-folded protein aggregates without biological activities. However, recent studies have revealed that some protein aggregates, known as non-classical or active IBs, contain correctly folded and functionally active proteins [13e15]. More recently, several self-assembling peptides have been used as the efficient inducer of active protein aggregates [16e18]. Compared with other aggregating fusion partners, self-assembling peptides are much
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
87
smaller in size and structurally simple, and have higher “pulldown” efficiencies. For example, self-assembling peptide ELK16 (LELELKLK)2 can spontaneously form b-sheet structure in aqueous solution, and function as an inducer of active protein aggregates when terminally attached to model proteins, including lipase A, amadoriase II, b-xylosidase and green fluorescent protein [17]. Therefore, self-assembling peptide-induced active aggregation is expected to have attractive application potentials in biotechnology, including in-situ immobilization of carrier-free enzymes, production of toxic proteins, and protein expression and purification [18]. In this study, we developed a simple method for recombinant protein purification by using self-assembling peptide ELK16-tagged TEVp (ELK16-TEVp). The proof of concept was provided by purifying recombinant bovine interferon-g (rBoIFN-g), which has been used to control many infectious cattle diseases [19e21]. We demonstrated that the ELK16-TEVp could be expressed as active protein aggregates and purified to a high purity with high protease activity.
ELK16-TEVp was purified using the method for active protein aggregate purification [17] with slight modification. For the pilot experiment, the expression of ELK16-TEVp was induced with 0.2 mM IPTG for 6 h at 30 C. Cells were harvested from 50 mL culture, and lyzed in 5 mL lysis buffer by sonication treatment. ELK16-TEVp was precipitated by centrifugation for 10 min at different centrifugation forces at 4 C. For the larger scale purification, the expression of ELK16-TEVp was induced for 16 h at optimized conditions. A total of 4 g wet weight cells was harvested from 500 mL culture and lyzed in 50 mL lysis buffer by sonication treatment. ELK16-TEVp was precipitated by centrifugation for 10 min at optimized centrifugation force at 4 C. After washing two times in 50 mL lysis buffer containing 0.5% Triton X-100, the purified ELK16-TEVp was washed one time with 50 mL TEVp cleavage buffer (50 mM Tris-HCl, pH8.0, 0.5 mM EDTA, 1 mM DTT), and dissolved in 5 mL TEVp cleavage buffer.
2. Materials and methods
2.5. Purification of His-BoIFN-g
2.1. Vector construction
His-BoIFN-g was purified using Ni-NTA Agarose Column (Qiagen, USA) under native conditions by following the product instruction. One milliliter of Ni-NTA slurry (50%) was loaded into a column (1 mL), and equilibrated with 8 mL lysis buffer. The column was loaded with 5 mL cleared bacterial lysate from 50 mL culture (0.95 g wet weight), and washed two times with 8 mL washing buffer containing 40 mM imidazole. His-BoIFN-g was eluted with 5 mL elution buffer containing 250 mM imidazole. The purified protein was dialyzed in 25 mL TEVp cleavage buffer for 9 h at 4 C with 3 buffer changes. After centrifugation for 10 min at 16,000g at 4 C, His-BoIFN-g was diluted to 500 mg/mL in TEVp cleavage buffer.
To construct an N-terminal self-assembling peptide fusion expression vector, the coding sequence for ELK16 peptide flanked by 17 proline (P) or threonine (T) rigid linkers [17] was adapted to E. coli codon usage using JAVA Codon Adaption Tool [22]. The synthetic sequence was cloned into pET-30a vector (Novagen, WI, USA) as a BglII/KpnI segment, and the resultant vector was called pET-P16P. To construct an ELK16-TEVp expression vector, the coding sequence for T17S/L56V/N68D/I77V/S135G quintuple mutant of TEVp [9] was adapted to E. coli codon usage, and the synthetic sequence was cloned into pET-P16P vector as an EcoRI/XhoI segment. The resultant vector was called pP16P-TEVp (Fig. 1a). To construct a His-BoIFN-g expression vector, the coding sequence for the mature peptide of BoIFN-g with a TEVp recognition sequence at the 50 end was amplified from pGEX-BoIFN-g vector [23] by PCR using rTaq DNA polymerase (TaKaRa, Dalian, China), and the forward primer FP (50 -GAGGATCCGAAAACCTGTACTTCCAGGGTCAGGGCCAATTTTTTAGAGAA-30 ) and reverse primer RP (50 -TACTCGAGTTACGTTGATGCTCTCCGGCC-30 ). The PCR product was cloned into pET-30a vector as a BamHI/XhoI segment, and the resultant vector was called pET-BoIFN-g (Fig. 2a). 2.2. Protein expression pP16P-TEVp or pET-BoIFN-g vector was transformed into E. coli strain BL21 (DE3), and the Luria broth culture supplemented with 50 mg/mL kanamycin was grown overnight at 37 C in an orbital shaker. The cell culture was diluted (1:100) in 2 YT medium (10 g yeast extract, 16 g tryptone, 5 g NaCl/L) supplemented with the same antibiotic, and grown to OD600 0.6 at 37 C. The expression of fusion protein was induced with different concentrations of IPTG for different times at different temperatures.
2.4. Purification of ELK16-TEVp
2.6. Protease cleavage and target protein recovery The protease cleavage reaction (100 mL) consisted of different amounts of ELK16-TEVp or GST-TEVp (BBI Life Sciences, USA) and His-BoIFN-g in the presence or absence of different additives. The cleavage reaction was carried out for 1 or 2 h at different pH at different temperatures. After cleavage, ELK16-TEVp was removed by centrifugation for 10 min at 16,000g at 4 C. Following the absorption with an equal volume of 50% Ni-NTA agarose slurry (Qiagen, USA) for 30 min at 4 C, His-tag was removed by an additional round of centrifugation. The cleavage reaction was followed by 12% or 15% SDS-PAGE analysis. After Commassie blue staining, the protein bands of interest were scanned using the Image Lab™ Software associated with GelDoc XR System (Bio-Rad, USA). The cleavage efficiency (%) was calculated according to the percent ratio of BoIFN-g/His-BoIFN-g band densities. One unit of ELK16-TEVp was defined as 85% cleavage of 3 mg substrate within 1 h at optimized conditions. The purified BoIFN-g was submitted to 1 cycle of Triton X-114 isothermal extraction to remove residual endotoxin [24]. The purified protein was mixed with 1% Triton X114 (final concentration), incubated for 10 min on ice and then 5 min at 37 C. After centrifugation for 1 min at 12,000g at 30 C, the upper aqueous phase was collected for antiviral assay.
2.3. Cell lysis 2.7. Antiviral assay After IPTG induction, cells were harvested by centrifugation for 10 min at 6,000g at 4 C, washed two times in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH7.2), and disrupted in the lysis buffer by sonication treatment (40 w, 10 s, 20 s intervals, 5 min). After centrifugation for 10 min at 6,000g (ELK16-TEVp) or 12,000g (His-BoIFN-g), the crude cell extract was collected for protein purification.
The antiviral activity of purified rBoIFN-g against vesicular stomatitis virus (VSV) was determined on MDBK cells (ATCC, USA) as described previously [25]. Briefly, the cells were seeded on 96-well plates, grown to 90% confluence and serial dilutions of rBoIFN-g were added in triplicates. After incubation for 24 h at 37 C, an optimal concentration (100 TCID50) of VSV was added. After
88
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
incubation for an additional 24 h, the antiviral activity was calculated according to the highest dilution of rBoIFN-g with 50% cytopathic inhibitory effect. 3. Results 3.1. Vector construction Since all of the reported recombinant TEV proteases are expressed with an N-terminal fusion tag, we constructed an Nterminal ELK16 fusion expression vector by cloning the peptide coding sequence downstream of the S-tag in pET-30a vector. Sequence analysis showed that the coding sequence for the quintuple TEVp mutant had an E. coli codon adaption index (CAI) of only 0.29. Therefore, the protease coding sequence was adapted to E. coli codon usage (CAI 1.0), and cloned in frame with the ELK16 coding sequence in the pET-P16P vector (Fig. 1a). To provide the proof of concept for protein purification using ELK16-TEVp, the coding sequence for BoIFN-g with a TEVp recognition sequence was cloned into pET-30a vector and expressed a His-BoIFN-g fusion protein (Fig. 2a). 3.2. Expression and purification of ELK16-TEVp Previous studies have shown that ELK16 fusion proteins can be expressed an active protein aggregates at 30 C. Therefore, we induced ELK16-TEVp expression first with different concentrations of IPTG at 30 C. SDS-PAGE analysis showed that, compared to the
Fig. 2. Expression and purification of His-BoIFN-g. (a) The schematic structure of HisBoIFN-g expression vector. (b) Induction of His-BoIFN-g expression with different concentrations of IPTG for 6 h at 37 C; (c) Purification of His-BoIFN-g. After induction with 0.1 mM IPTG for 16 h at 20 C, cells were harvested from 250 mL culture, and HisBoIFN-g was purified using Ni-NTA column under native conditions. The protein samples were analyzed on 12% SDS-PAGE. Lane 1: total cell extract; lane 2: insoluble fraction; lane 3: soluble fraction; lane 4: purified His-BoIFN-g.
uninduced cell lysate, an expected 37-kDa extra protein band (5.5kDa PT-ELK16-PT þ 26.3-kDa TEVp þ 5.2-kDa vector sequence) was present in IPTG-induced cell lysate, and 0.2 mM IPTG was sufficient to induce the high level expression (Fig. 1b). About 90% of ELK16TEVp could be precipitated by centrifugation force as low as 1,000g, the amount of which was increased slightly as the increase of centrifugation force (Fig. 1c). Since more than 90% of ELK16-TEVp could be precipitated by lower centrifugation forces, the fusion protein was precipitated at a larger scale (500 mL culture) by centrifugation at 6,000g. After washing two times in 0.5% Triton X100 and one time in TEVp cleavage buffer, quantitative SDS-PAGE analysis showed that ELK16-TEVp was purified to 91% purity with 92% recovery (Fig. 1d). 3.3. Expression and purification of His-BoIFN-g
Fig. 1. Expression and purification of ELK16-TEVp. (a) The schematic structure ELK16TEVp expression vector. (b) Induction of ELK16-TEVp expression at 30 C with different concentrations of IPTG; (c) Precipitation of ELK16-TEVp at different centrifugation forces; (d) Purification of ELK16-TEVp. After induction with 0.2 mM IPTG for 16 h at 20 C, cells were harvested from 500 mL cell culture, and ELK16-TEVp was precipitated by centrifugation at 6,000g, washed two times in 0.5% Triton X-100 and one time in TEVp cleavage buffer. The protein samples were analyzed on 12% SDS-PAGE. Lane 1: total cell extract; lane 2: soluble fraction; lane 3: insoluble fraction; lane 4: protein aggregates washed one time in Triton X-100; lane 5: protein aggregates washed two times in Triton X-100; lane 6: protein aggregates washed one more time in TEVp cleavage buffer.
First, the expression of His-BoIFN-g was induced with different concentrations of IPTG for 6 h at 37 C. SDS-PAGE analysis showed that, compared to the uninduced cell lysate, an expected extra 23kDa protein band (5.4-kDa His- and S-tags þ 16-kDa target protein) was revealed in the IPTG-induced cell lysate, 50% of which was present in the insoluble fraction. The highest level expression could be induced with 0.1 mM IPTG (Fig. 2b). Then, the expression of HisBoIFN-g was induced with 0.1 mM IPTG for 16 h at 20 C, and purified from 250 mL culture using Ni-NTA Agarose column under the native condition. Quantitative SDS-PAGE analysis showed that HisBoIFN-g was purified to 87.5% purity with a recovery of 88.0% (Fig. 2c). 3.4. Optimization of the temperature for active ELK16-TEVp expression Since high temperature is one of the main reasons for classic
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
89
with the highest cleavage rate (93%) at pH8.0 at 37 C (Fig. 4a). In consideration of the potential detrimental effect of higher temperature (37 C) on the protease stability, the following cleavage reaction was performed at 30 C. Quantitative SDS-PAGE analysis showed that the cleavage efficiency of ELK16-TEVp was also pHdependent with the highest cleavage rate (91%) at pH8.0 (Fig. 4b). 3.6. Determination and comparison ELK16-TEVp activity
Fig. 3. Optimization of the temperature for active ELK16-TEVp expression. The expression of ELK16-TEVp was induced with 0.2 mM IPTG for 6 h at different temperatures. The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of purified ELK16-TEVp for 1 h at H8.0 at 30 C. The cleavage reactions were analyzed on 12% SDS-PAGE, and the band densities of BoIFN-g and His-BoIFN-g were scanned for the calculation of cleavage rates after Coomassie blue staining.
inclusion body formation, we optimized the temperature for more active ELK16-TEVp expression. First, the expression of ELK16-TEVp was induced at different temperatures and purified as described. Then, 500 ng purified ELK16-TEVp was used to cleave 30 mg HisBoIFN-g under the standard conditions (pH8.0, 30 C, 1 h). Quantitative SDS-PAGE analysis showed that the cleavage efficiency was decreased as the increase of ELK16-TEVp expression temperature, with the highest cleavage rate (64%) at expression temperature of 20 C (Fig. 3). Therefore, the following experiments were performed using the ELK16-TEVp expressed at 20 C.
First, ELK16-TEVp activity was determined by cleaving a fixed amount (3 mg) of His-BoIFN-g with different amounts of ELK16TEVp under standard conditions. Quantitative SDS-PAGE analysis showed that the cleavage efficiency was ELK16-TEVp dosedependent with a cleavage rate of almost 100% at 150 ng. By using 85% cleavage of 3 mg substrate for the unit (U) definition, ELK16-TEVp activity was calculated to be 1U/75 ng or 1.3 104 U/ mg protein (Fig. 5a). Then, the same amount (4 U) of ELK16- or GSTTEVp was used to cleave 30 mg His-BoIFN-g under standard conditions. Quantitative SDS-PAGE analysis showed that ELK16-TEVp had a cleavage rate of 84% (1 h) or almost 100% (2 h), which was much higher than that (30% or 88%) of GST-TEVp (Fig. 5b). 3.7. Effects of additives on ELK16-TEVp activity It is generally accepted that high concentrations of salts, elutants (e.g., imidazole) and detergents (e.g., urea and SDS) in protein purification buffers can affect enzyme activities, including TEVp. To detect the effects of different additives on ELK16-TEVp activity, 30 mg His-BoIFN-g was cleaved with 500 ng EKL16-TEVp in the presence of different additives. Quantitative SDS-PAGE analysis showed that the cleavage rate was enhanced slightly in the presence of 50 mM NaCl, but inhibited significantly by higher concentration (250 mM) of NaCl (Fig. 6a). Unlike the relatively stable activity in the presence of different concentrations of imidazole (Fig. 6b), ELK16-TEVp activity was inhibited completely by higher concentrations of urea (2 M) or SDS (0.5%) (Fig. 6c and d). 3.8. Purification of rBoIFN-g using ELK16-TEVp
3.5. Optimization of the conditions for ELK16-TEVp cleavage To optimize the conditions for substrate cleavage, 30 mg HisBoIFN-g was cleaved with 500 ng ELK16-TEVp for 1 h at different pH at different temperatures. Quantitative SDS-PAGE analysis showed that the cleavage efficiency was temperature-dependent
To provide the proof of concept for protein purification using ELK16-TEVp, 30 mg His-BoIFN-g was cleaved with 500 ng EKL16TEVp under standard conditions. SDS-PAGE analysis shows that His-BoIFN-g was cleaved efficiently, and ELK16-TEVp was removed completely from the cleavage reaction by centrifugation. After
Fig. 4. Optimization of the conditions for ELK16-TEVp cleavage. (a) The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of ELK16-TEVp for 1 h at pH8.0 at different temperatures; (b) His-BoIFN-g was cleaved with ELK16-TEVp for 1 h at different pH at 30 C. The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
90
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
Fig. 5. Comparison of ELK16-TEVp activity with GST-TEVp. (a) The same amount (3 mg) of His-BoIFN-g was cleaved with different amounts of ELK16-TEVp for 1 h at pH8.0 at 30 C. The dashed lines indicate the amount of ELK16-TEVp required for 85% cleavage of 3 mg His-BoIFN-g; (b) His-BoIFN-g was cleaved with the same amount (4 U) of ELK16- TEVp or GSTTEVp for 1 or 2 h at pH8.0 at 30 C. The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
Fig. 6. Effects of additives on ELK16-TEVp activity. The same amount (30 mg) of His-BoIFN-g was cleaved with the same amount (500 ng) of ELK16-TEVp for 1 h at pH 8.0 at 30 C in the presence of different concentrations of NaCl (a), imidazole (b), urea (c) or SDS (d). The cleavage reactions were analyzed by quantitative SDS-PAGE as described.
absorption with Ni-NTA beads and an additional round of centrifugation, rBoIFN-g was purified to 98.3% purity with 63% recovery (Fig. 7). The performance of ELK16-TEVp for rBoIFN-g purification is summarized in Table 1.
3.9. Antiviral activity of rBoIFN-g After isothermal extraction with Triton X-114, cytopathic inhibition assay showed that the purified rBoIFN-g had an antiviral activity of 1.6 103 U/mg protein against VSV without overt cytotoxicity (Table 2).
4. Discussion TEVp is a popular protease for recombinant protein purification due to its high cleavage efficiency and stringent sequence specificity. Although several recombinant TEV proteases have been Table 1 Performance of purification of rBoIFN-g using ELK16-TEVp. Purification step
Fig. 7. Purification of rBoIFN-g using ELK16-TEVp. A total amount (30 mg) of His-BoIFNg was cleaved with 500 ng ELK16-TEVp for 1 h at pH8.0 at 30 C. After cleavage, ELK16TEVp was removed by centrifugation at 16,000g, and the released His-tag was removed by absorption with Ni-NTA and an additional centrifugation. The protein samples were analyzed on 15% SDS-PAGE. Lane 1: His-BoIFN-g before cleavage; lane 2: cleavage product before ELK16-TEVp removal; lane 3: cleavage product after ELK16-TEVp removal; lane 4: purified rBoIFN-g after His-tag removal.
ELK16-TEVp Cell extract Centrifugation (pellet) Triton X-100 wash Cleavage buffer wash His-BoIFN-g Cell extract Affinity purification After tag cleavage Purified BoIFN-g
Purity (%)
Recovery (%)
Yield (mg/L cell culture)a
26.6 72.3 86.6 91.0
100 95 94 92
132 125 124 121
35.1 87.5 57.6 98.3
100 88 79 63
80 70 63 50
a A total of 8.0 or 9.5 g wet weight cells/L culture was used for purification of ELK16-TEVp or His-BoIFN-g.
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
91
Table 2 Antiviral activity of rBoIFN-g purified using ELK16-TEVp. Group
VSV challenge
rBoIFN-g
100 TCID50
Positive controlb Negative controlc
100 TCID50
a b c
Triplication
1 2 3 þ
CPE50 with indicated dilution of rBoIFN-ga 21
22
23
24
25
26
27
28
29
210
þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
The concentration of stock rBoIFN-g solution was 0.25 mg/mL. The positive control represents the cells were infected with VSV in the absence of rBoIFN-g. The negative control represents the cells were treated with rBoIFN-g only.
reported, most of them are expressed as His- or GST-tagged proteins which require affinity columns for purification and/or removing from the cleavage reactions [10e12]. Recent studies have shown that some self-assembling peptides can act as the inducer of active protein aggregates and their fusion proteins can be purified by centrifugation with high “pull-down” efficiencies [16e18]. In this study, we developed a simple method for recombinant protein purification by using ELK16-TEVp and provided the proof of concept by purification of rBoIFN-g. Among the four self-assembling peptides used in protein purification, all of them are used as a C-terminal fusion tag [16e18]. However, all of the reported TEV proteases are expressed with an Nterminal fusion tag [10e12]. In our pilot experiment, we constructed an N-terminal ELK16 fusion expression vector by cloning the peptide coding sequence into pET-30a vector as an NdeI and BglII segment, but failed to express ELK16-TEVp under all conditions tested. Since the peptide coding sequence was only 51-bp away from the ATG codon in pET-30a vector, we speculated that the fusion protein translation may be interrupted by the proximal N-terminal secondary structure formed by ELK16, which is a selfcomplementary b peptide. To confirm this hypothesis, in this study we separated ELK16 coding sequence with two PT linkers, and cloned the coding sequence more distant (204 bp) from the translation start codon in pET30a vector. Our expression experiment showed that ELK16-TEVp was expressed not only efficiently, but formed active protein aggregates as well. Our experimental data suggest the following advantageous use of ELK16-TEVp for recombinant protein purification. First, ELK16TEVp could be expressed efficiently in E. coli, the expression level (132 mg/L culture) of which was higher than that of other fusiontagged TEVp [7,12]. This may be contributed the factor that the ELK16-TEVp was a quintuple mutant which has improved expression level, solubility and/or stability [9,10]. In addition, the higher expression level may be due to the use of E. coli codon-adapted TEVp coding sequence for expression. Unlike the wild type TEVp that can cleave itself to produce truncated enzymes with diminished activities [6], only single ELK16-TEVp protein band was detected before and after purification, confirming the resistance of quintuple TEVp mutant to auto-activation [6,8,9]. Second, ELK16TEVp could be purified to high purity (91%) with high recovery (92%) by centrifugation due to its expression as active protease aggregates. The first indication for this was the sticky nature of the recombinant bacterial extract. The second evidence was that ELK16-TEVp could be precipitated by centrifugation force as low as 1,000g, which was much lower than that (12,000g) for precipitation of classic inclusion bodies. The precipitation of ELK16-TEVp was neither salt- nor temperature-dependent since it could be precipitated at 4 C or room temperature in buffers with or without 50 mM NaCl. The purification process could be accomplished within 1 h without the need of expensive agents and special equipment. Third, ELK16-TEVp was highly active with almost 100%
cleavage efficiency (2 h) which was significantly higher than that (88%) of GST-TEVp. This may be explained by the simple structure and small size of ELK16 peptide [16], which was favorable for TEVp expression and/or folding. More importantly, unlike other fusiontagged TEVp which requires an extra affinity purification step to remove from the cleavage reaction, ELK16-TEVp could be removed from the cleavage reaction by centrifugation without the need of buffer change. The Ni-NTA absorption step used in this study was to remove the His-tag, but not ELK16-TEVp. Further studies are under the way to improve the purification system by expressing the target proteins as ELK16 fusions. If successful, both ELK16-TEVp and cleaved ELK16-tag could be removed from the cleavage reactions by single-step centrifugation. Finally, by using ELK16-TEVp, rBoIFN-g could be purified to high purity (98.3%) with high recovery (63%), which was similar or higher than that of chromatographic purification methods [19e21]. One of several advantages of TEVp is its adaptability to different buffer conditions. In this study, for example, ELK16-TEVp activity remained relatively stable at temperatures ranging from 4 C to 40 C, and at pH ranging from 6.5 to 9.5, which was similar to other TEV proteases. Interestingly, ELK16-TEVp activity was enhanced slightly by a low concentration (50 mM) of NaCl, but inhibited significantly by higher concentrations (100 mM) of NaCl. ELK16TEVp activity was not affected in the presence of different concentrations of imidazole, indicating its needless to remove from the protein elutants for ELK16-TEVp cleavage. Finally, ELK16-TEVp activity was affected significantly by higher concentrations of urea (2 M) or SDS (0.5%), which was different from MBP- or Histagged TEVp [10]. 5. Conclusions In this paper we report a novel method for recombinant protein purification using self-assembling peptide ELK16-TEVp. Since its expression as active protein aggregates, ELK16-TEVp could be purified to a high purity and removed from the cleavage reaction by centrifugation. The proof of concept was provided by purifying rBoIFN-g. Our data suggest ELK16-TEVp may become a universal tool for recombinant protein purification. Acknowledgments This work was supported by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and the fund from Jiangsu Key Laboratory of Zoonosis. References [1] A. Malhotra, Tagging for protein expression, Methods Enzymol. 463 (2009) 239e258. [2] D. Walls, S.T. Loughran, Tagging recombinant proteins to enhance solubility and aid purification, Methods Mol. Biol. 681 (2011) 151e175.
92
G.-Y. Li et al. / Protein Expression and Purification 128 (2016) 86e92
[3] D.S. Waugh, An overview of enzymatic reagents for the removal of affinity tags, Protein Expr. Purif. 80 (2011) 283e293. [4] J.A. Daros, M.C. Schaad, J.C. Carrington, Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants, J. Virol. 73 (1999) 8732e8740. [5] R.B. Kapust, J. Tozser, T.D. Copeland, D.S. Waugh, The P1' specificity of tobacco etch virus protease, Biochem. Biophys. Res. Commun. 294 (2002) 949e955. [6] R.B. Kapust, J. Tozer, J.D. Fox, D.E. Anderson, S. Cherry, T.D. Copeland, D.S. Waugh, Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency, Protein Eng. 14 (2001) 993e1000. [7] S. van den Berg, P.A. Lofdahl, T. Hard, H. Berglund, Improved solubility of TEV protease by directed evolution, J. Biotechnol. 121 (2006) 291e298. [8] L.D. Cabrita, D. Gilis, A.L. Robertson, Y. Dehouck, M. Rooman, S.P. Bottomley, Enhancing the stability and solubility of TEV protease using in silico design, Protein Sci. 16 (2007) 2360e2367. [9] L. Wei, X. Cai, Z. Qi, L. Rong, B. Cheng, J. Fan, In vivo and in vitro characterization of TEV protease, Protein Expr. Purif. 83 (2012) 157e163. [10] C. Sun, J. Liang, R. Shi, X. Gao, R. Zhang, F. Hong, Q. Yuan, S. Wang, Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives, Protein Expr. Purif. 82 (2012) 226e231. [11] K. Zhu, X. Zhou, Y. Yan, H. Mo, Y. Xie, B. Cheng, J. Fan, Cleavage of fusion proteins on the affinity resins using the TEV protease variant, Protein Expr. Purif. (2016) 30023e30027. S1046-5928. [12] X.J. Li, J.L. Liu, D.S. Gao, W.Y. Wan, X. Yang, Y.T. Li, H.T. Chang, L. Chen, C.Q. Wang, J. Zhao, Single-step affinity and cost-effective purification of recombinant proteins using the sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner, Protein Expr. Purif. 119 (2016) 51e56. [13] E. Garcia-Fruitos, N. Gonzalez-Montalban, M. Morell, A. Vera, R.M. Ferraz, A. Aris, S. Ventura, A. Villaverde, Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins, Microb. Cell Fact. 4 (2005) 27. [14] S. Jevsevar, V. Gaberc-Porekar, I. Fonda, B. Podobnik, J. Grdadolnik, V. Menart,
[15] [16]
[17]
[18] [19] [20]
[21]
[22]
[23] [24]
[25]
Production of nonclassical inclusion bodies from which correctly folded protein can be extracted, Biotechnol. Prog. 21 (2005) 632e639. J.P. Arie, M. Miot, N. Sassoon, J.M. Betton, Formation of active inclusion bodies in the periplasm of Escherichia coli, Mol. Microbiol. 62 (2005) 427e437. X. Wang, B. Zhou, W. Hu, Q. Zhao, Z. Lin, Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8, Microb. Cell Fact. 14 (2015) 88. W. Wu, L. Xing, B. Zhou, Z. Lin, Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli, Microb. Cell Fact. 15 (2011) 9. B. Zhou, L. Xing, W. Wu, X.E. Zhang, Z. Lin, Small surfactant-like peptides can drive soluble proteins into active aggregates, Microb. Cell Fact. 11 (2012) 10. T. Kashima, A. Morishita, H. Iwata, K. Maeda, T. Inoue, Expression of bovine cytokines in Escherichia coli, J. Vet. Med. Sci. 61 (1999) 171e173. K. Murakami, A. Uchiyama, T. Kokuho, Y. Mori, H. Sentsui, T. Yada, M. Tanigawa, A. Kuwano, H. Nagaya, S. Ishiyama, H. Kaki, Y. Yokomizo, S. Inumaru, Production of biologically active recombinant bovine interferongamma by two different baculovirus gene expression systems using insect cells and silkworm larvae, Cytokine 13 (2001) 18e24. D.N. Wedlock, E.E. Doolin, N.A. Parlane, S.J. Lacy-Hulbert, M.W. Woolford, B.M. Buddle, Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils, J. Dairy Res. 67 (2000) 189e197. A. Grote, K. Hiller, M. Scheer, R. Munch, B. Nortemann, D.C. Hempel, D. Jahn, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host, Nucl. Acids Res. 33 (2005) 526e531. J.J. Xu, A.J. Qin, Y.L. Liu, W.J. Jin, R.F. Li, Cloning of cDNA for bovine interferon-g and its expression in E. coli, J. Yangzhou Univ. 24 (2003) 5e10 (In Chinese). R. Ma, J. Zhao, H.C. Du, S. Tian, L.W. Li, Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction, Anal. Biochem. 424 (2012) 124e126. B.L. Osborn, H.S. Olsen, B. Nardelli, J.H. Murray, J.X. Zhou, A. Garcia, G. Moody, L.S. Zaritskaya, C. Sung, Pharmacokinetic and pharmacodynamic studies of a human serum albumin -interferon-alpha fusion protein in cynomolgus monkeys, J. Pharmacol. Exp. Ther. 303 (2002) 540e548.